Enzymes which digest proteins are widely distributed in nature. Both intracellular and extracellular proteases exist in a wide variety of organisms. Extracellular proteases are produced by micro-organisms to enable them to convert proteinaceous material to low molecular weight peptides for transport into the cell, to satisfy either carbon and energy requirements and/or nitrogen requirements for growth. Bacteria, fungi and yeasts are know to produce different proteases, both intracellular and extracellular, whose biochemical characteristics and properties have been described; see e.g. The Enzymes, Ed. Paul D. Boyer Vol. III "Hydrolysis--peptide bonds". 3rd Edition 1971.
We have now found it possible to produce extracellular proteases from M. sedentarius which are capable of solubilizing human callus material and degrading other proteinaceous material.
According to one aspect of the present invention there are provided enzyme materials isolated from a culture of M. sedentarius which comprises one or more proteases having an ability to degrade protein including human callus.
According to another aspect of the present invention there is provided an enzyme material having the following characteristics:
water-soluble, PA1 non-dialysable (through a membrane having a molecular weight cut-off of 10 kDa), PA1 an isoelectric point of 4.6, PA1 a molecular weight of 30.3 kDa, PA1 an optimum pH for protease activity at about 8.2, PA1 an optimum temperature at about 40.degree. C. PA1 water-soluble, PA1 non-dialysable (through a membrane having a molecular weight PA1 cut-off of 10 kDa), PA1 an isoelectric point of 2.7, PA1 a molecular weight of 50 kDa; PA1 an optimum pH for protease activity at about pH 10.2, PA1 an optimum temperature at about 46.degree. C.
and
The enzyme material which may be obtained from M. sedentarius, has been found to have at 35.degree. C., protease activity in the pH range of about 5.1--about 10.9, and human callus degrading activity at 40.degree. C. in the pH range of about 5.9--about 8.0. The enzyme material has been found to have an optimum pH for human callus degrading activity at about 7.1 and an optimum temperature at about 40.degree. C.
According to a further aspect of the present invention there is provided an enzyme material having the following characteristics:
and
This enzyme material, which also may be obtained from M. sedentarius has been found to have at 35.degree. C., protease activity in the pH range of about 5.1--about 11.3, and human callus degrading activity at 50.degree. C. in the range of about 4.3--about 10.0. The enzyme material has been found to have an optimum pH for callus degrading of about 7.5 and an optimum temperature at about 50.degree. C.
According to another aspect of the present invention there is provided a process for the preparation of enzyme materials of the present invention comprising isolating the enzyme materials from a culture of M. sedentarius.
According to another aspect of the invention there is provided a cell-free culture of M. sedentarius which exhibits protease activity.
In all the above aspects, it is preferred to employ the novel strain of M. sedentarius (NCIMB No. 40287) described below.
According to other aspects of the present invention there are provided processes for degrading human callus or corns comprising the application of an enzyme material or cell-free culture of the present invention to human callus or corns.
According to another aspect of the present invention there is provided a process for degrading protein comprising the application of an enzyme material or cell-free culture of the present invention to protein.
According to another aspect of the present invention there is provided a process for the preparation of degradation product from proteinaceous material comprising the application of an enzyme material of the present invention to proteinaceous material.
According to a further aspect of the present invention there is provided a strain of M. sedentarius deposited under The Budapest Treaty at the National Collections of Industrial and Marine Bacteria on May 24, 1990 under the accession number NCIMB 40287.
Preferably, in a protein degradation or digestion process of the present invention the pure enzyme material, a formulation thereof, or a cell-free culture of M. sedentarius is applied to the material to be treated.
In one preferred embodiment the enzyme materials of the present invention are isolated from the strain M. sedentarius NCIMB 40287.
The enzyme materials of the second and third aspects of the present invention are hereinafter referred to as Protease 1 and Protease 2; these proteases having been obtained by culturing M. sedentarius NCIMB 40287.
Other strains of M. sedentarius will produce proteases which are similar to Protease 1 and 2, insofar as their biological activities are concerned. Such other proteases are intended to be within the scope of the invention.